Conventionally, as a method for detecting biomolecules such as nucleic acid, protein, and enzyme, a fluorescence measurement method utilizing fluorescence reaction has been adopted. Since the fluorescence measurement method can perform measurement of biomolecules safely and inexpensively by combining existing optical components such as a light source and a light receiving unit without using a radioisotope, it is applied to various kinds of biomolecule detection methods such as enzyme immunoassay, electrophoresis, and confocal scanning fluorescence microscopy.
The fluorescence measurement is a method for detecting a fluorescence signal emitted from a sample by irradiating the sample with excitation light. For example, FITC (fluorescein isothiocyanate) is a substance that emits fluorescence having a wavelength of 520 nm when it is irradiated with excitation light having a wavelength of 495 nm. In order to detect a substance that emits fluorescence, the fluorescence is measured by a combination of light having an excitation wavelength and a light receiving unit for detecting a fluorescence wavelength. As a practical application thereof, a fluorescence measurement method is proposed in which excitation light irradiates a sample, and the fluorescence depending on the excitation light is detected by a light receiving unit disposed on the irradiation side of the excitation light (for example, refer to Japanese Published Examined Patent Application No. Hei. 6-60901 (Patent Document 1)). FIG. 10 shows a schematic diagram of a conventional fluorescence measurement apparatus. With reference to FIG. 10, excitation light e1, which is emitted from an excitation light source 103 and reflected by a dichroic mirror 107, irradiates a sample 106 disposed on a substrate 101. Fluorescence f1 emitted from the sample 106 that is excited by the excitation light e1 is transmitted through the dichroic mirror 107 and a bandpass filter 108 to be detected by the light receiving unit 104 as a fluorescence signal. In this way, various kinds of fluorescent samples and fluorescently-labeled biomolecules can be detected by using a substance that emits fluorescence, and excitation light and a light receiving unit corresponding to the fluorescence.
When performing a fluorescence measurement, a substrate, a cell, a channel or the like for holding a sample is used (hereinafter referred to simply as “substrate”). As a material of the substrate, silica glass having a high transparency to ultraviolet light has conventionally been adopted. However, recently, high-polymer materials that are easily moldable and disposable have been used. Although, as described above, such high-polymer materials are easily moldable, they tend to emit autofluorescence when irradiated with excitation light. Since autofluorescence from the substrate 101 has a similar wavelength region to that of the fluorescence f1, the autofluorescence transmits through the dichroic mirror 107 and the bandpass filter 108 to reach the light receiving unit 104. The autofluorescence causes background noise, thereby worsening the S/N ratio of the measurement. Accordingly, various methods for reducing autofluorescence from the substrate 101 have conventionally been proposed (refer to Japanese Published Patent Application No. 2000-338035 (Patent Document 2), Japanese Published Patent Application No. 2002-286627 (Patent Document 3), Japanese Published Examined Patent Application No. Hei. 6-95073 (Patent Document 4), Japanese Published Patent Application No. 2003-130873 (Patent Document 5), and Japanese Published Patent Application No. 2003-183425 (Patent Document 6)).
For example, Patent Document 2 proposes a method of performing spectrofluorometric measurement. A fluorescence wavelength from a sample is slightly different from an autofluorescence wavelength from a substrate, whereby fluorescence can be separated from autofluorescence. Patent Document 3 proposes a method of reducing autofluorescence from a substrate by covering a part of the substrate other than a fluorescence measurement part with a light shielding film. Further, Patent Document 4 proposes a method for preventing emission of autofluorescence from a substrate by reflecting excitation light with a reflection layer such as a metal layer or a dielectric multilayer that is disposed on the surface of the substrate where a fluorescent substance is disposed. Furthermore, Patent Document 5 and Patent Document 6 propose a method of making a material of a substrate so as not to emit autofluorescence.
Furthermore, also when using fluorescence measurement for measuring a sample that performs electrophoresis, the above-mentioned fluorescence measurement method is adopted (for example, refer to Japanese Patent No. 2624655 (Patent Document 7)). Also in this case, as described above, autofluorescence emitted from a substrate as a channel causes background noise during fluorescence measurement, thereby worsening detection sensitivity.
In order to solve this problem, Japanese Published Patent Application No. 2004-279306 (Patent Document 8) proposes a method in which a region other than a measurement part is covered with a light shielding part, and excitation light irradiates a side surface of an electrophoresis gel cassette as a substrate, thereby preventing the electrophoresis gel cassette from emitting autofluorescence.
As described above, a technique for detecting a small signal of sample with high sensitivity has recently been demanded. Therefore, various methods have been developed for reducing influences of autofluorescence emitted from a substrate, leakage of excitation light, and background noise such as scattered light, in a technique of irradiating a sample with excitation light and analyzing light emitted from the sample.
However, the above-mentioned respective methods for reducing influence of autofluorescence from a substrate have the following drawbacks.
In the method of separating autofluorescence from a fluorescence signal by spectrofluorometric measurement, which is proposed by Patent Document 2, since the wavelength region of the autofluorescence and the wavelength region of the fluorescence from the sample are approximately equal to each other or broadly overlap each other, it is difficult to completely separate them by spectrofluorometric measurement. Therefore, the autofluorescence signal is undesirably added over the fluorescence signal. Especially when measuring a sample having a small fluorescence signal, a fluorescence signal is difficult to detect because of an autofluorescence signal, which makes it difficult to detect a small fluorescence of sample with high sensitivity.
Further, in the method of reducing autofluorescence from a substrate by covering a part of a substrate other than a fluorescence measurement part, which is proposed by Patent Document 3, autofluorescence undesirable occurs from a bottom of a sample holder. Therefore, when measuring a sample emitting a small fluorescence signal, a fluorescence signal is difficult to detect because of an autofluorescence signal, which makes it difficult to perform highly sensitive detection. Moreover, thin film fabrication processes for the light shielding film are complicated.
Further, in the method of preventing autofluorescence from a substrate by reflecting excitation light using a reflection layer comprising a metal or a dielectric multilayer, which is disposed on the surface of the substrate on which a fluorescent substance is disposed, which method is proposed by Patent Document 4, since a dichroic mirror and a light receiving filter have a transmittance limit of 10˜10−6% ((leakage light intensity/incident light intensity)×100(%)), it is difficult to completely cut the reflected excitation light, and the leakage light of the excitation light from the filter causes a noise signal. Especially when measuring a sample having a small fluorescence signal, the noise signal significantly deteriorates the S/N ratio.
Further, in the method for making a material itself of a substrate so as not to emit autofluorescence, which is proposed by Patent Documents 5 and 6, it is difficult to completely prevent the material from emitting autofluorescence, and therefore, a small amount of autofluorescence is emitted from the substrate. Especially when measuring a sample having a small fluorescence signal, the autofluorescence significantly deteriorates the S/N ratio.
Further, in the method of irradiating excitation light onto a side surface of an electrophoresis gel cassette by fluorescence measurement in electrophoresis, when irradiating the excitation light onto the side surface of the electrophoresis gel cassette, it is difficult to irradiate the excitation light to the entire gel evenly, and moreover, a complicated optical control for irradiating the excitation light onto the side surface of the electrophoresis gel cassette is required. Therefore, this method is not practical.
As described above, there have conventionally been proposed various methods for reducing the influence of autofluorescence. However, none of these methods has sufficient performance for detecting a small amount of sample signal.